Atherosclerosis remains a major health problem in the U.S., with significant morbidity and mortality. Imaging of the arteries allows the clinician to screen, detect and characterize the disease to provide useful, anatomical information which has been utilized to guide decisions about treatment and to enable the delivery of therapy in the case of percutaneous interventions (Holmes et al., Am Heart J. 1983; 106:981-8; Kennedy et al., Circulation. 1982; 66:III16-23; Serruys et al., N Engl J Med. 2001; 344:1117-24). However, x-ray angiography is an invasive procedure, and it images only the vessel lumen and hence the silhouette of the subset of lesions that impinge upon the lumen. Atherosclerosis can develop in the arterial wall and be accommodated by outward (or positive) arterial remodeling (Glagov et al., N Engl J Med. 1987; 316:1371-5; Ward et al., Circulation. 2000; 102:1186-91). The importance of this phenomenon has been highlighted in angiographic studies demonstrating that non-severe stenoses are more often associated with acute coronary events than are severe coronary stenoses (Ambrose et al., J Am Coll Cardiol. 1988; 12:56-62; Little et al., Circulation. 1988; 78:1157-66). From a pathological perspective, plaques with large lipid cores and thin fibrous caps are more prone to rupture, leading to thrombosis and vascular events, than plaques with small, securely contained lipid cores and thick caps (Falk et al., Circulation. 1995; 92:657-71; Fuster et al., Lancet. 1999; 353 Suppl 2:SII5-9).
To date, numerous different techniques have been employed to image characterize and analyze atherosclerotic plaques. Such techniques include angioscopy, thermography, near-infrared spectroscopy, near-infrared photon tomography, optical coherence tomography, Raman spectroscopy, computed tomography, nuclear imaging, and ultrasound techniques (Fayad et al., Circ Res. 2001; 89:305-16; Saijo et al., Atherosclerosis. 2001; 158:289-95; Chen et al., Circulation. 2002; 105:2766-71; Tsimikas et al., Am J Cardiol. 2002; 90:22L-27L; Lindner, Am J Cardiol. 2002; 90:32L-35L; Madjid et al., Am J Cardiol. 2002; 90:36L-39L; Fayad et al., Circulation. 2002; 106:2026-34; Fayad et al., Neuroimaging Clin N Am. 2002; 12:351-64). However magnetic resonance imaging (MRI) has emerged as the preferred technique for atherosclerosis plaque imaging. MRI is a non-invasive, non-destructive, three-dimensional imaging technique that differentiates tissue structure on the basis of proton magnetic resonance properties with a wide range of image contrast (Choudhury et al., J Cardiovasc Risk. 2002; 9:263-70; Choudhury et al., Arterioscler Thromb Vasc Biol. 2002; 22:1065-74). The non-invasive nature of MRI allows virtually unlimited number of repetitive measurements to be made in a single animal, and permits time-based assessment of a given plaque's lesion size and other morphological features (i.e., progression, regression, or compositional changes).
Athersclerotic lesions from a variety of large animals have previously been examined using MRI systems (e.g., rabbits (Skinner et al., Nature Medicine. 1995; 1:69-73; McConnell et al., Arterioscler Thromb Vasc Biol. 1999; 19:1956-9; Worthley et al., Circulation. 2000; 101:586-9; Worthley et al., Circulation. 2000; 102:II-809; Johnstone et al., Arterioscler Thromb Vasc Biol. 2001; 21:1556-60; Helft et al., J Am Coll Cardiol. 2001; 37:1149-1154; Helft et al., Circulation. 2002; 105:993-8), pigs (Lin et al., J of Magn Reson Imaging. 1997; 7:183-90; Worthley et al., Circulation. 2000; 101:2956-2961; Corti et al., J Am Coll Cardiol. 2002; 39:1366-1373), non-human primates (Kaneko et al., Circulation. 1996; 94:I-346. Abstract), and humans (Toussaint et al., Circulation. 1996; 94:932-8; Yuan et al., Circulation. 1998; 98:2666-71; Coulden et al., Heart. 2000; 83:188-91; Hatsukami et al., Circulation. 2000; 102:959-64; Fayad et al., Circulation. 2000; 101:2503-2509; Fayad et al., Circulation. 2000; 102:506-510; Botnar et al., Circulation. 2000; 102:2582-7; Corti et al., Circulation. 2001; 104:249-52; Yuan et al., Radiology. 2001; 221:285-99; Yuan et al., Clin N Am. 2002; 12:391-401; Jaffer et al., Arterioscler Thromb Vasc Biol. 2002; 22:849-54; Ouhlous et al., J Magn Reson Imaging. 2002; 15:344-51; Fayad et al., Neuroimaging Clin N Am. 2002; 12:461-71; Kim et al., Circulation. 2002; 106:296-9; Corti et al., Circulation. 2002; 106:2884-7; Cai et al., Circulation. 2002; 106:1368-73) by use of conventional MR systems (1.5T) with a spatial resolution ≧300 μm. To study small structures, such as the aorta of mice (less ˜1 mm in luminal diameter), it is necessary to increase the signal-to-noise ratio by use of high-magnetic field equipped with small radiofrequency (RF) coils. The strong magnetic fields allow the use of MR microscopy (MRM) with in vivo spatial resolution of 25-100 μm (Johnson et al., Magnetic Resonance Quarterly. 1993; 9:1-30; Weissleder, Nature Rev Cancer. 2002; 2:11-8).
Non-contrast enhanced MRM plaque imaging and characterization techniques utilize the inherent differences in “natural” MR relaxation properties of different plaque components. Recent reports show that contrast-enhanced gadolinium-MRI may be useful for atherosclerotic plaque characterization such as differentiation between fibrotic and non-fibrotic plaques, identification of neovasculature, and possible detection of plaque inflammatory status (Aoki et al., Radiology. 1995; 194:477-81; Aoki et al., J Magn Reson Imaging. 1999; 9:420-7; Weiss et al., J. Magn. Reson. Imaging. 2001; 14:698-704; Yuan et al., J Magn Reson Imaging. 2002; 15:62-7; Wasserman et al., Radiology. 2002; 223:566-73). The gadolinium (Gd) contrast agents, routinely used in clinical MRI, alter the MR proton relaxation of the imaged tissue. The chelated Gd does not penetrate phospholipid cellular membranes because of its highly hydrophilic properties (Merbach et al., The chemistry of contrast agents in medical magnetic resonance imaging. Chischester: John Wiley & Sons; 2001). It stays entirely confined into the extracellular space after intravenous administration, does not bind plasma proteins and it is eliminated unmetabolized by the kidneys (Niendorf et al., Safety and risk of Gadolinium-DTPA: extended clinical experience after more than 20 million applications. Berlin: Blackwell Wissenschafts-Verlag GmbH; 1996). This non-specific enhancement to the plaque and/or plaque components may facilitate the imaging and the characterization of mouse lesions.
However, in order to achieve good resolution of the MR image, a certain quantity of the imaging agent must accumulate at the site of interest being examined. Preferably, the imaging agent should specifically accumulate at the site being examined. For example, the required tissue concentration of an MR contrast agent is ˜10−4-10−6 Molar (Nunn et al., Q J Nucl Med. 1997; 41:155-62; Aime et al., J Magn Reson Imaging. 2002; 16:394-406). For radionuclide imaging it is only ˜10−10 Molar. This is a great challenge since the molecular epitopes expressed at 10−9 or 10−12 molar concentrations must be detected. Another challenge is to get the imaging agent to and into the site of interest. One way to reach the required local concentration of an MR contrast agent is to use nanoparticulate carriers, such as micelles, emulsions, or liposomes, which carry the imaging agent to the site of interest. However, the size of these liposomes and emulsions is such that it exceeds the size required to readily permeate into the extracellular space and hence into a plaque (Sloop et al., J Lipid Res. 1987; 28:225-37). For example, liposomes typically have a diameter of about 100-400 nm and cannot enter a plaque unless the endothelium is damaged (e.g., Lanza et al., Circulation. 2002; 106:2842-7; Li et al., Radiology. 2001; 218:670-8). Therefore, delivery of imaging agents through the use of such nanoparticles is practically restricted to either targets on the endothelium or in lesions in which endothelial integrity has been breached, for example, as after balloon angioplasty (Lanza et al., Circulation. 2002; 106:2842-7).
Reconstituted lipoproteins have previously been used as delivery vehicles for lipophilic drugs. Lipoproteins are produced mainly by the intestine and liver (or by processing of intestine or liver-derived lipoproteins) and are the native transporters in the circulation of a variety of lipophilic and hydrophilic compounds and are classified into 4 main categories depending on size and composition (i.e., in order of decreasing diameter: chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) (Havel et al., The Metabolic and Molecular Bases of Inherited Disease. New York: McGraw-Hill; 2001:2705-2716). With the exception of HDLs, the lipoproteins also suffer the same drawbacks as micelles, conventional emulsions and liposomes, in that the entities are too large to serve as good vehicles for the delivery of imaging agents.
LDLs are particularly unsuitable for such delivery because, in addition to being larger than the optimal size (on average LDLs are larger than 20 nm), the major protein constituent of LDLs is apoB, a very large and hydrophobic protein, which makes it difficult to reconstitute LDL (rLDL) particles. Further, LDL moieties are spontaneously retained in atherosclerotic lesions (Williams et al., Arterioscler Thromb Vasc Biol. 1995; 15:551-61), thereby making it difficult to selectively detect specific molecular targets of interest within the plaque. Yet another factor that makes LDLs unattractive as delivery vehicles is that LDL is an atherogenic particle, and so it is difficult to justify the possible risks from administration of rLDL to patients already at high risk for cardiovascular disease.
Micelles, much like LDLs, also do not serve well as delivery vehicles to enter atherosclerotic plaques, because they are spontaneously retained for prolonged periods of time, rendering them unsuitable for the selective detection of specific molecules of interest.
Hence, there is a need for a delivery vehicle reconstituted that is small enough to freely enter an atherosclerotic plaque and that is not readily trapped unless specifically modified and provides sufficient quantities of an imaging agent for use in MRI or MR spectroscopy or other imaging techniques such as CT, gamma-scintigraphy, and optical, positron emission tomography (PET), and combined imaging techniques.